The elucidation of intracellular trafficking pathways is a major challenge in cell biology and promises to lead to significant new insights into basic cellular processes. The advances in single molecule methodologies hold the expectation that the trafficking mechanisms for proteins/macromolecules can be unraveled not only for bulk populations, but even at the level of individual molecules. Due to the photo-stability of quantum dots (QDs) it is possible to observe QD-labeled molecules for extended observation periods, which is necessary to follow intracellular pathways of individual molecules. However, current microscopy modalities are not well suited to address problems related to intracellular trafficking in three dimensions. Whereas classical microscopes image one focal plane at a time, cells are three dimensional objects and the trafficking pathways are not typically restricted to one focal plane. This, combined with the fact that the dynamics are often very fast, means that detailed trafficking studies are often not possible since the pathways cannot be captured. A further significant problem is the low depth discrimination capability of a classical microscope. This means that from an image it is very difficult to determine the three dimensional position of an object such as a QD-labeled single molecule. This makes it highly problematic to study in detail processes such as the pathway from endocytosis to early endosome or the pathway from sorting endosome to exocytosis. To address these problems we have recently developed a new imaging modality with which different focal planes can be imaged at the same time. This modality includes the capability to image events at the plasma membrane with total internal reflection fluorescence microscopy, whilst simultaneously capturing processes in the interior of the cell using epifluorescence mode in higher focal planes. Using this approach QD-labeled proteins can be imaged as they follow a three dimensional pathway through a cell. Equally important is the promise that the depth discrimination problem can be overcome with this approach. A central aspect of this project will be to develop algorithms with which the three dimensional location of a single molecule can be identified. The proposed approaches will be tested on an important trafficking problem, i.e. the intracellular trafficking, endocytosis and exocytosis of QD-labeled immunoglobulin G molecules. Our Specific aims are: 1. To develop analytical tools to determine the 3D location of a QD-labeled protein in a cell. 2. To analyze how accurately the 3D position of a QD labeled protein can be determined. 3. To test the proposed algorithms on experimental data. PUBLIC HEALTH RELEVANCE: We propose to develop a new technology for the imaging of living cells. This technology promises to overcome significant limitations in existing approaches which have to date prevented researchers from studying central aspects of the functioning of cells. The new technology will permit investigations that are important to increase our understanding of how cells function. Significantly, this technology will also improve the tools that researchers have available to investigate how a rapidly expanding class of therapeutics, namely antibody-based drugs, interact with cells.